rK5-induced autophagy is Beclin 1 dependent. (A) Representative immunoprecipitation of Beclin 1 in rK5-treated HUVECs. Cell lysates (200 μg/mL) collected at different time points were immunoprecipitated by anti–Beclin 1 antibody and blotted with anti–Beclin 1 antibody. (B) Confocal microscopy images of HUVECs showing endogenous Beclin 1 levels at different time points after rK5 treatment. Nuclei were labeled by DAPI. Beclin 1 expression (red) was detected using an anti–Beclin 1 monoclonal antibody. Alexa Fluor 647 goat anti–mouse IgG was used as secondary antibody. rK5-treated HUVEC (16 hours) sample without the primary antibody treatment was used as a negative control. (C) HUVECs were grown to subconfluent conditions. Scrambled shRNA or shRNA specific to Beclin 1 were transfected using Lipofectamine 2000. Top row shows total-cell lysates of untransfected HUVECs (lane 1), scrambled shRNA-transfected HUVECs (lane 2), and shRNA specific to Beclin 1–transfected HUVECs (lane 3) that were analyzed to determine Beclin 1 levels by Western blot. Bottom row shows confocal images of shRNA-transfected HUVECs showing Beclin 1 levels (red) 36 hours after transfection. Nuclei were stained with DAPI (blue). (D) HUVECs were cotransfected with LC3-GFP (green) and shRNA. Transfected HUVECs were cultured in 5% FBS in M199 with or without 40 ng/mL VEGF. Scrambled shRNA and shRNA specific to Beclin 1–transfected cells were exposed to 1.5 μg/mL rK5 for 24 hours. Treated HUVECs were labeled with Mitotracker Red. Confocal microscopy images were obtained at 600× magnification.